Chronic metabolic acidosis induces a series of homeostatic responses that are designed to return blood pH to normal values. In the renal proximal tubule, this response includes increases in hydrogen ion secretion, ammonia synthesis and citrate absorption. Studies from this laboratory have examined the molecular basis by which these adaptations occur, focusing on regulation of the apical membrane Na/H antiporter, NHE3. During the last grant period, we showed that significant roles are played by the endothelins, Pyk2, c-Src, ERK, and focal adhesion proteins. The proposed studies continue our focus on the mechanisms by which the proximal tubule senses a decrease in intracellular pH and relays the signals so as to eventually increase NHE3 activity. Studies are divided into four aims. In aim 1, studies will utilize microarrays to identify novel acid-regulated and endothelin-regulated genes. In aim 2 we will focus on nocturnin. Preliminary results with microarrays have identified nocturnin as the most highly regulated gene in acute acidosis. Nocturnin is a circadian rhythm gene. Studies will examine the relationship between circadian rhythm and renal acidification and the role of nocturnin in acid signaling in the proximal tubule. In aim 3 we will study the endothelin B receptor. Previous studies have demonstrated that the endothelin B receptor can activate NHE3 and that it plays a key role in activation of NHE3 by acid. Two domains within the endothelin B receptor have been found to be key, the C-terminal tail and the second intracellular loop (ICL2). Studies will utilize the yeast two-hybid system to identify proteins that bind to ICL2. Using the yeast two-hybrid system, we have shown that Aldolase B binds to the C-terminal domain, and will now study its role in regulating NHE3. Lastly, studies will be performed to determine the specific role played by the endothelin B receptor in acid signaling. The last aim will study Pyk2. Pyk2 is activated in acidosis and we have shown that it is directly regulated by ambient pH, indicating that it can serve as a pH sensor. Studies will determine the specific domains/amino acids responsible for its pH sensitivity, and define the role that Pyk2 plays in the acid signaling cascade.